The specific aim of this research program is to study the mechanisms of regulation of oncofetal gene expression in hepatocellular carcinogenesis in the mouse liver. Alpha-Feto-protein is oncofetal antigen whose synthesis is activated in the adult liver by 3'-methyl-4-dimethylaminoazobenzene (DAB). Studies will be done to determine whether the repressed AFP gene of the adult liver undergoes structural changes as a process of its activation. In the first part of this program the temporal patterns of activation of AFP synthesis will be established, i.e., to determine whether there is a primary phase of AFP synthesis, followed by a period of repression and then a second increase in AFP synthesis. Serum AFP levels and AFP synthesis will be determined by radioimmunoassay; AFP-mRNA levels will be determined by cDNA-mRNA reassociation kinetics and the appearance of the primary transcript and processed intermediates will be determined by DMB paper hybridization techniques. Studies will also be done to determine whether DNA and chromatin modifications occur in the AFP structural gene during DAB-activation. Differences in methylation of cytosine residues in the AFP-structural gene and its flanking regions will be determined by analyses with methylation sensitive and insensitive restriction enzymes. Chromatin modifications will be determined by the sensitivity of AFP-gene to DNase I and micrococcal nuclease digestion. Attempts will be made to determine differences in non-histone chromatin proteins (NHCP) by protein blotting techniques. Fractionation of the NHCP which are involved in AFP-gene activation and repression will be attempted. AFP-cDNA clones will be prepared in the plasmid pBR322, and genomic clones will be selected from a mouse genomic library. These cloned sequences will be used as a source of probes for these studies.